探討ClC-2、LPS、TNFα於小鼠睪丸萊氏細胞中對睪固酮生成基因表現之影響

Abstract

睪丸為精子生成與睪固酮合成的重要器官，主要有曲精細管和間質，而睪固酮是由間質的萊氏細胞所合成。睪固酮對男性生殖系統的發育與精子生成的調控扮演了關鍵角色。睪固酮的合成由LH透過cAMP調控，cAMP會促進類固醇生成基因Star、Cyp11a1、Cyp17a1、Hsd3b1等的表現，進而促進睪固酮的生成。氯離子通道蛋白2 (Chloride channel-2, ClC-2) 為第一型氯離子通道蛋白家族中的一員，廣泛分布於人體各個組織器官中，扮演了調節細胞內外離子濃度的重要角色。先前研究發現ClC-2缺乏使小鼠無法正常產生精子，導致公鼠不孕。臨床也有ClC-2突變的男性患者發現無精症狀的案例，說明ClC-2對精子生成具有重要性，但ClC-2在睪丸的生理功能尚不清楚。本篇論文利用小鼠萊氏腫瘤細胞株MA-10以及睪丸分離的初代萊氏細胞探討ClC-2在睪固酮生成中可能扮演的角色。此外，已知男性不孕症患者中有5 ~ 10% 病例的病因和發炎反應相關，有研究指出LPS與其下游的促發炎因子TNFα會抑制小鼠睪固酮生成。本篇論文亦以MA-10以及萊氏細胞的模式，分析LPS、TNFα對睪固酮生成相關基因表現的影響。 西方墨點法結果顯示，在MA-10細胞與萊氏細胞中都可以偵測到ClC-2蛋白質的表現。在MA-10中將ClC-2 knockdown會使Cyp11a1以及Hsd3b1的mRNA和蛋白質量減少。MA-10給予ClC-2抑制劑GaTx2處理，只觀察到在有8-br-cAMP情況下Cyp11a1蛋白質量減少；在萊氏細胞則使Cyp17a1 mRNA量下降，另外在有8-br-cAMP情況下會使Star mRNA增加。此外，我們還分析ClC-2促進劑lubiprostone的效應，在MA-10有8-br-cAMP情況下會增加Hsd3b1蛋白質的量，另外也使Cyp17a1蛋白質增加，但卻造成Cyp11a1 mRNA減少；在萊氏細胞則都沒有效應。由於這些處理沒有得到一致性結論，因此ClC-2在睪固酮生成所扮演的角色有待更多的研究。 LPS處理萊氏細胞使Star mRNA及Hsd3b1蛋白質量減少；另外在有8-br-cAMP情況下會使Cyp17a1蛋白質量下降，但在MA-10則是Cyp17a1 mRNA減少。TNFα的處理會使MA-10與萊氏細胞中大部分睪固酮生成相關基因表現量降低，其中以Cyp11a1、Hsd3b1最明顯。這些結果說明TNFα可經由抑制睪固酮生成基因的表現而影響睪固酮的合成。

Testis is the organ for spermatogenesis and testosterone production. It is mainly comprised of seminiferous tubules and interstitium. Testosterone is synthesized by Leydig cell in the interstitium. Testosterone plays a crucial role in reproductive system development and spermatogenesis. Testosterone synthesis is regulated by LH through cAMP that stimulates the expression of steroidogenic genes including Star, Cyp11a1, Cyp17a1, Hsd3b1, and thereby promotes the production of testosterone. Chloride channel protein 2 (ClC-2) is a member of the class I ClC family, which is widely presented in various tissues and organs in human. It plays an important role in regulating ion concentration between intracellular and extracellular. Previous studies found that lacking ClC-2 prevented mice from producing sperm normally which caused male mice infertility. A clinical case report indicated that a male patient with ClC-2 mutation had azoospermia. These studies indicate the importance of ClC-2 in spermatogenesis. However, the physiological function of ClC-2 in testis remains unknown. In this study we used mice tumor Leydig cells MA-10 and primary Leydig cells isolated from the testis to investigate the role of ClC-2 in testosterone biosynthesis. Moreover, it is known that 5 ~ 10% of male infertility patients have an etiology related to inflammation. LPS and its downstream pro-inflammatory cytokine TNFα has been shown to inhibit mice testosterone synthesis. Therefore, we also analyzed the effect of LPS and TNFα on the expression of genes involved in testosterone synthesis. Western blot results revealed that ClC-2 proteins were present in both MA-10 and Leydig cells. ClC-2 knockdown led to a decrease in the mRNA and protein levels of Cyp11a1 and Hsd3b1 in MA-10. MA-10 was further treated with ClC-2 inhibitor GaTx2 and the results showed that only Cyp11a1 protein level was reduced in the presence of 8-br-cAMP. In Leydig cell, GaTx2 reduced Cyp17a1 mRNA but increased Star mRNA in the presence of 8-br-cAMP. In addition, treatment of MA-10 cell ClC-2 activator lubiprostone resulted in the increase of Cyp17a1 protein level and also Hsd3b1 protein in the presence of 8-br-cAMP; however, the Cyp11a1 mRNA was decreased by lubiprostone. Lubiprostone had no significant effects on Leydig cells. Because there are no consistent results from these treatments, the role of ClC-2 in the synthesis of testosterone needs further studies. Our data also showed that TNFα, but not LPS, had significantly inhibitory effect on the expression of steroidogenic genes in MA-10 and Leydig cells. The results suggest that TNFα may affect testosterone production by inhibiting the expression of genes involved in testosterone synthesis.